Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) are believed to initiate cell transformation by formation of covalent DNA adducts. Mammalian metabolism of the hydrocarbon gives two diastereomeric DEs, each of which exists as a pair of enantiomers, in which the benzylic hydroxyl group and epoxide oxygen are either cis or trans. BaP DE adducts described in the present report are derived from the two enantiomers of the latter diastereomer. Adducts formed in DNA by these DEs result from cis and trans opening of the epoxide ring by the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. We have developed powerful new synthetic methods to prepare site- and stereospecific DE-DNA adducts for use in probing the structural biology and mechanistic enzymology of enzymes required for DNA processing, such as HIV-1 integrase, helicases, topoisomerases and DNA polymerases. Studies during the past year have focused on topoisomerases, which relax torsional strain in DNA by making transient single- or double-strand breaks that permit the passage of single or double stranded DNA, respectively, through the break, followed by rejoining of the cleaved ends. Topoisomerases play critical roles in DNA replication, transcription and recombination and are targets for widely used antibacterial drugs such as ciprofloxacin and antitumor drugs such as camptothecin. Several mechanistically distinct families of topoisomerases have been characterized in viral, procaryotic and eucaryotic systems. Both human topoisomerase (top1) and vaccinia topoisomerase function by making reversible single-strand breaks in DNA to give a phosphotyrosyl bond between the enzyme and a DNA 3'-phosphate group at the cleavage site, with expulsion of a DNA fragment containing a free 5'-hydroxyl group. We have previously reported that minor-groove bound dG adducts derived from trans opening of BaP DE, when located at or near a cleavage site for human top1, inhibit cleavage at this site and induce new cleavages at several positions remote from the adduct. We have now examined the effects of these dG adducts on vaccinia virus topoisomerase, which unlike the human enzyme, is highly specific for cleavage at a 5'-CCCTT-XXX target sequence and does not exhibit any cleavage when this site is blocked. We have mapped interactions between the minor groove and the active site of vaccinia topoisomerase by measuring the effect of site- and stereospecific BaP DE-dG adducts on the rates of the cleavage step. The cleavage rate (relative to unadducted control) in the presence of a dG adduct on the non-scissile strand was relatively unaffected when the adduct was at the position immediately 3' (downstream) to a base flanking the cleavage site or 4 bases 5' (upstream) from the site. In contrast, nearly 2000-fold inhibition was observed with dG adducts 2 or 3 bases upstream from the cleavage site. Thus a specific region of the DNA substrate was identified where minor-groove interactions with the enzyme are critical. We have also examined site-specific interactions vaccinia topoisomerase with BaP DE-dA adducts, which intercalate either 5' or 3' to the modified adenine when the configuration at the point of attachment to N-6 of dA is R or S, respectively. When the hydrocarbon is intercalated between the two bases flanking the cleaved (T-X) bond or between the immediately adjacent bases on the 5'-side of the site, the cleavage rate is dramatically retarded (2500-16,000-fold). This retardation decreases markedly when intercalated hydrocarbon is placed farther away from the cleaved bond. Unlike human top1 and vaccinia, human topoisomerase II alpha (top2) is a homodimeric enzyme that makes two breaks, staggered by four base pairs, in double-stranded DNA. Intercalated BaP DE-dA adducts inhibit top2 cleavage when they are located either outside the four base-pair stagger or in the middle of the stagger. However, when the hydrocarbon is intercalated at or immediately adjacent to either of the staggered cleavage sites, the DNA is cleaved, but the resultant top2-DNA covalent complex is trapped by inhibition of religation, thus poisoning the enzyme.